Pawelczyk T, Olson M S
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
Biochem J. 1992 Dec 1;288 ( Pt 2)(Pt 2):369-73. doi: 10.1042/bj2880369.
The activity of pyruvate dehydrogenase (PDH) kinase in the purified PDH complex from pig kidney is sensitive to changes in ionic strength. The enzyme has optimum activity within a small range of ionic strength (0.03-0.05 M). An increase in ionic strength from 0.04 M to 0.2 M lowers the activity of PDH kinase by 32% and decreases the Km for ATP from 25 microM to 10 microM. At constant ionic strength (0.15 M) the enzyme has optimum activity over a broad pH range (7.2-8.0). The PDH kinase is stimulated 2.2-fold by 20 mM-K+, whereas Na+ even at high concentration (80 mM) has no effect on the enzyme activity. The stimulation of PDH kinase by K+ is not dependent on pH and ionic strength. PDH kinase is inhibited by HPO4(2-) in the presence of K+, whereas HPO4(2-) has no effect on the activity of this enzyme in the absence of K+. HPO4(2-) at concentrations of 2 and 10 mM inhibits PDH kinase by 28% and 55% respectively. The magnitude of this inhibition is not dependent on the ATP/ADP ratio. Inhibition by HPO4(2-) in the concentration range 0-10 mM is non-competitive with respect to ATP, and becomes mixed-type at concentrations over 10 mM. The Ki for HPO4(2-) is 10 mM. When HPO4(2-) is replaced by SO4(2-), the same effects on the activity of PDH kinase are observed. PDH kinase is also inhibited by Cl-. In the presence of 80 mM-Cl- the PDH kinase is inhibited by 40%. The inhibition by Cl- is not dependent on K+. In conclusion, we postulate that changes in phosphate concentrations may play a significant role in the regulation of PDH kinase activity in vivo.
猪肾纯化丙酮酸脱氢酶（PDH）复合体中丙酮酸脱氢酶激酶的活性对离子强度的变化敏感。该酶在较小的离子强度范围内（0.03 - 0.05M）具有最佳活性。离子强度从0.04M增加到0.2M会使PDH激酶的活性降低32%，并使ATP的Km值从25μM降至10μM。在恒定离子强度（0.15M）下，该酶在较宽的pH范围内（7.2 - 8.0）具有最佳活性。20mM - K⁺可使PDH激酶的活性提高2.2倍，而即使Na⁺浓度很高（80mM）对该酶的活性也没有影响。K⁺对PDH激酶的刺激作用不依赖于pH和离子强度。在有K⁺存在时，HPO₄²⁻会抑制PDH激酶，而在没有K⁺时，HPO₄²⁻对该酶的活性没有影响。2mM和10mM浓度的HPO₄²⁻分别使PDH激酶的活性抑制28%和55%。这种抑制的程度不依赖于ATP/ADP比值。在0 - 10mM浓度范围内，HPO₄²⁻对ATP而言是非竞争性抑制，而在浓度超过10mM时变为混合型抑制。HPO₄²⁻的Ki值为10mM。当用SO₄²⁻取代HPO₄²⁻时，观察到对PDH激酶活性有相同的影响。Cl⁻也会抑制PDH激酶。在80mM - Cl⁻存在时，PDH激酶被抑制40%。Cl⁻的抑制作用不依赖于K⁺。总之，我们推测磷酸盐浓度的变化可能在体内PDH激酶活性的调节中起重要作用。
